S140: SINGLE-CELL MULTIOMICS ANALYSES REVEAL COMPLEX INTRA-PATIENT HETEROGENEITY IN RELAPSED CLL FOLLOWING VENETOCLAX THERAPY
Authors: R. Thijssen, L. Tian, C. Flensburg, M. A. Anderson, A. Jarratt, H. Peng, I. Majewski, C. Tam, J. Seymour, P. Blombery, M. Ritchie, D. Huang, A. Roberts
Journal: HemaSphere (2022)

Background: Venetoclax (VEN) is the first approved-in-class BH3-mimetic therapy that inhibits pro-survival BCL2 to induce apoptosis. It is now a standard of care for patients with chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia. Despite high remission rates, secondary resistance remains problematic with disease progressing on continuous therapy in most patients. While previous studies by ourselves and others demonstrated a BCL2 mutation (Blombery et al., 2019 Cancer Disc) or MCL1 amplification (Guièze et al., 2019 Cancer Cell) confer resistance in patient samples, it is clear that these only account for a fraction of tumour cells at disease progression and are not present in all leukaemias. Given that these changes only provide partial explanations, other mechanisms must operate to subvert the action of VEN. Aims: To uncover why VEN fails in patients by assessing heterogeneity, distinguishing cells with known changes (e.g., BCL2 mutation, MCL1 amplification) and to elucidate what is happening in the cells not harbouring these alterations. Methods: We applied a single-cell (sc) sequencing method that simultaneously measures gene expression and full-length transcripts to determine genotype on samples from patients with progressive CLL with long term follow-up on trials of continuous VEN therapy (Figure 1). Results: By applying scRNA-seq to 161,499 tumour cells, we discovered a high degree of inter- and intra-patient heterogeneity. The majority of CLL cells displayed an altered transcriptional profile at relapse compared to before VEN treatment. 8/13 VEN-relapse samples demonstrated a complex sub-clonal architecture (Figure 1). By applying scLong-read sequencing, we detected BCL2 mutations in 4/13 samples at relapse. We also identified subclones with loss of NOXA or BAX, but overall, alterations in genes for the pro-apoptotic proteins were uncommon. In contrast, altered expression of the pro-survival genes was common and high MCL1 expression was seen in most of the CLL cells in 11/13 relapses. This could only be fully explained by MCL1 amplification in 1 patient and partially in 2 (Figure 1). At VEN resistance, we identified near universal activation of NF-ΚB in circulating tumour cells and this was highly correlated with MCL1 expression. We next demonstrated that the MCL1 locus was transcriptionally targeted by NF-ΚB accounting for the MCL1 increase. NF-ΚB activation and associated high MCL1 was also observed in cells harbouring the BCL2 mutations. Strikingly, NF-ΚB activation (and consequent increased MCL1) dissipated after VEN cessation and commencement of a BTK inhibitor as the leukaemic cells substantially recovered their in vitro sensitivity to VEN. Consistent with the hypothesis that VEN was driving these changes, NF-ΚB activation (and high MCL1) was not detected in a patient (CLL26) with disease relapse 3 years after ceasing VEN in CR. The disease in this patient responded promptly to VEN reinduction. Image:Summary/Conclusion: Taken together, our findings provide a much clearer understanding of resistance to VEN and provide impetus for improved VEN-based clinical management of CLL patients. Given the multiple ways a CLL population in a patient can become resistant to ongoing VEN, it seems unlikely that simply adding other drugs at relapse will be durably effective in most patients. The data pinpoint NF-ΚB activation as a biomarker for in vivo VEN-resistance and provide a specific biological rationale for ceasing VEN in deep response, as is currently being used in time-limited and explored in response-directed regimens.